The Effect of LncRNA OIP5-AS1 on Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Targeting miR-126-5p/TRAF3 Axis

This study aims to explore the effect of LncRNA (long non coding RNA) OIP5-AS1 (Opa interacting protein 5 antisense RNA 1) on hypoxia/reoxygenation (H/R)-induced myocardial cell injury by tar geting the miR-126-5p (microRNA-126-5p)/TRAF3 (tumor necrosis factor receptor associated factor 3) axis. To validate the interaction relationships between LncRNA OIP5-AS1 and miR-126-5p, as well as between miR 126-5p and TRAF3, this study employed a dual-luciferase reporter assay. Different treatment groups were con structed, and H9C2 cells were divided into the following 10 groups: ① NC group (normal culture); ② H/R group (cultured in glucose-free medium placed in an incubator with 94% N₂, 5% CO₂, and 1% O₂ for 2 hours of hypoxia, then the medium was replaced with sugar-containing medium and cultured under 95% O₂ and 5% CO₂ for 24 hours); ③ OE-NC group (H/R treatment+transfection with empty vector control); ④ OE-OIP5-AS1 group (H/R treatment+transfection with OIP5-AS1 overexpression vector); ⑤ OE-OIP5-AS1+mimic NC group (H/R treatment+co-transfection with OIP5-AS1 overexpression vector and mimic NC); ⑥ OE-OIP5-AS1+miR-126-5p mimic group (H/R treatment+co-transfection with OE-OIP5-AS1 and miR-126-5p mimic); ⑦ sh-NC group (H/ R treatment+transfection with sh-NC); ⑧ sh-OIP5-AS1 group (H/R treatment+transfection with sh-OIP5-AS1); ⑨ sh-OIP5-AS1+anti-NC group (H/R treatment+co-transfection with sh-OIP5-AS1 and anti-NC); ⑩ sh-OIP5 AS1+anti-miR-126-5p group (H/R treatment+co-transfection with sh-OIP5-AS1 and anti-miR-126-5p). MTT was performed to measure H9C2 cell proliferation. Flow cytometry was performed to measure apoptosis in H9C2 cells. ELISA method was used to measure the expression of SOD, ROS, and MDA in H9C2 cells. Western blot was used to detect the expression of TRAF3 and Cleaved Caspase-3/Caspase-3 proteins in H9C2 cells. The experimental result shows, LncRNA OIP5-AS1 was able to target and negatively regulate miR-126-5p. MiR-126-5p was able to target and negatively regulate TRAF3. In H9C2 cells subjected to H/R treatment, the expression of LncRNA OIP5 AS1, TRAF3 mRNA, and protein increased, while the expression of miR-126-5p decreased. The D490 value was reduced, accompanied by significant oxidative stress (evidenced by elevated ROS and MDA levels, and decreased SOD activity). The apoptosis rate and the expression of the apoptosis-related protein Cleaved Caspase-3/Caspase-3 were also increased (P<0.01). Overexpression of OIP5-AS1 further upregulated the mRNA, and protein expression of LncRNA OIP5-AS1, TRAF3 in H/R-treated H9C2 cells, downregulated miR-126-5p expression, and reduced the D490 value (P<0.01). Conversely, overexpression of miR-126-5p mitigated the inhibitory effect of OIP5-AS1 overexpression on the proliferation of H/R-treated H9C2 cells (P<0.01). Knockdown of OIP5-AS1 decreased the expression of LncRNA OIP5-AS1, TRAF3 mRNA and protein, promoted miR-126-5p expression, increased the D490 value, alleviated oxidative stress (reduced ROS and MDA, increased SOD), and lowered the apoptosis rate and Cleaved Caspase-3/Caspase-3 expression in H/R-treated H9C2 cells (P<0.01)). Inhibition of miR-126-5p reversed the ameliorative effects of OIP5-AS1 knockdown on H/R-treated H9C2 cells (P<0.01). Knockdown of LncRNA OIP5-AS1 may alleviate H/R-induced myocardial cell injury by targeting the miR-126-5p/TRAF3 axis.

CSTR: 32200.14.cjcb.2026.03.0014